In animal and in the first human studies, our group showed that AAV2-CFTR could be applied to the nasal epithelium, maxillary sinus, and right lower lobe of the human lung without any adverse affects. These studies and multiple animal studies published in the literature showed that genes expressed from AAV vectors result in persistent expression in a variety of tissues. A recent multi institutional collaborative study showed statistically significant improvement in FEV1 at Day 30 and induced sputum IL-8 levels observed between Days 14-45 following 3 aerosolized doses of 1 x 10[13] DNAse resistant particles tgAAVCF. Thus, AAV vectors have great potential as therapeutic agents. Despite these promising studies several hurdles must be overcome before AAV vectors will be useful therapeutic agents. This grant will use a combination of animal and human studies to address the hypothesis that development of a new serotype with new more powerful promoters delivered to the airways efficiently via a bronchoscopic Microsprayer will be safe and result in increased levels of recombinant gene expression. There are three overall questions that will be addressed. 1. Will Dosing with a pseudotyped vector based on the AAV5 virus lead to increased expression from the recombinant vector? Experiments in this specific aim will be directed specifically toward addressing, if delivery of AAV5-CFTR to the mammalian airway elicits an immunologic or inflammatory response and if so to what aspect of the vector is the immunologic response directed. 2. Will inclusion of a more powerful promoter augment CFTR expression from recombinant vectors? Thus, we will explore the potential risks and benefits of using an altered CFTR construct and a new chicken beta-actin promoter to enhance expression of CFTR. It is anticipated that answers to these questions in a non-human primate model will provide the preclinical data necessary to launch a study of aerosolized AAV5-CFTR vector in patients with CF. 3. Does aerosol delivery of new higher titer AAV5-CFTR vectors administered to CF patients with Mild Lung Disease lead to uniform gene transfer and CFTR expression? Particular attention will also be given to whether the new pseudotyped vector AAV5-CFTR in humans leads to increased expression of the recombinant vector. It is expected that answers to this question will lead to a practical delivery system for recombinant AAV5-CFTR with potential new promoters to boost CFTR expression.